Physical exercise is a powerful stimulator of glucose uptake and metabolism in skeletal muscle and thus is an important regulator of glucose homeostasis in both normal and pathophysiological states. The overall goal of this research project is to determine the signaling mechanisms through which physical exercise stimulates glucose uptake and metabolism, and increases the sensitivity of these metabolic processes to insulin. To achieve this goal two strategies will be taken; one will take advantage of the fact that physical exercise and insulin are similar metabolic stimulators and thus study the effects of exercise on identified steps in the insulin signaling pathway, the other will use exercise- stimulated glucose transporter translocation as a starting point for the elucidation of "upstream" signaling events. Initial experiments have been designed to determine the effects of exercise on early tyrosine phosphorylation steps in the insulin signaling pathway. For this purpose, skeletal muscle insulin receptor and insulin- receptor substrate-1 (IRS-1) tyrosine phosphorylation will be studied in vivo following muscle contractions and following insulin administration after muscle contractions. The next set of experiments will determine if exercise alters the phosphatidylinositol 3-kinase (PI 3-kinase) cell signaling pathway, the mechanism of this putative alteration, and the role this system may play in the effects of exercise to increase glucose uptake and insulin sensitivity. For this purpose, the effects of exercise, insulin, and insulin in the post-exercise state on the activity and subcellular localization of PI 3-kinase will be measured. To investigate the effects of insulin and exercise on other putative post-receptor signaling steps in the insulin action pathway in skeletal muscle, the effects of exercise and insulin on tyrosine phosphorylation of mitogen activating protein kinase (MAP kinase) in vivo and MAP kinase activity will be measured. Additional experiments are designed to establish a link between contraction- and insulin-activated cell signaling molecules and the glucose transport system by investigating the subcellular distribution and physical association of signaling proteins with the glucose transporter system. For these studies, subcellular fractionation and immunoprecipitation methods will be used to determine if IRS-1, PI 3- kinase, MAP kinase, or other signaling intermediaries co-localize or are physically associated with glucose transporters in skeletal muscle intracellular microsomal membranes or plasma membranes. These studies will begin to define the molecular mechanisms of contraction-stimulated changes in skeletal muscle glucose uptake and metabolism. Ultimately, these studies should provide us with a better understanding of glucose regulation during exercise in normal individuals and in individuals with diabetes mellitus.